1

Performance evaluation of the Novodiag® Bacterial GE+ multiplex PCR assay 1

Charly Roy1, David Robert

4, Lucie Bruhl

2,3, Alice Buissonnière

2,3, Astrid Ducournau

2,3, 2

Francis Mégraud2,3

, Emilie Bessède2,3

, Delphine Boraud4, Philippe Lehours

2,3*.

3

4

1-University Hospital Pellegrin, Bordeaux, France; 5

2-French National Reference Center for Campylobacters & Helicobacters, Hôpital Pellegrin, 6

33076 Bordeaux, France; 7

3-INSERM, Univ. Bordeaux, UMR1053 Bordeaux Research In Translational Oncology, 8

BaRITOn, 33000 Bordeaux, France; 9

4-EXALAB, Labexa group, Le Haillan, France. 10

11

*Corresponding author: Prof Philippe Lehours, INSERM, Univ. Bordeaux, UMR1053 12

Bordeaux Research In Translational Oncology, BaRITOn, 146 rue Léo Saignat, 33000 13

Bordeaux, France. Tel:+33 5 57 57 12 86; mail: philippe.lehours@u-bordeaux.fr 14

15

Running title: Performance Evaluation of the Novodiag® Bacterial GE+ 16

Keywords: Novodiag® Bacterial GE+, syndromic panel-based, analytical performance, 17

digestive infections. 18

JCM Accepted Manuscript Posted Online 22 July 2020J. Clin. Microbiol. doi:10.1128/JCM.01033-20Copyright © 2020 American Society for Microbiology. All Rights Reserved.

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Abstract 19

The bacteriological diagnosis of intestinal bacterial infections has historically been based on 20

culture on agar plates. However, culture may lack sensitivity and some enteropathogens such 21

as pathovars of Escherichia coli may escape routine diagnosis. Our goal was to evaluate the 22

analytical performance of the Novodiag® Bacterial GE+ kit, for the detection of 23

enteropathogenic bacteria in acute community diarrhea. 24

251 stools were included in this study (198 retrospective and 53 prospective). The analytical 25

performance was calculated using a composite reference standard (CRS) in absence of a 26

perfect gold standard (lack of sensitivity of culture). The CRS was defined as positive if 27

culture was positive or, in case of a negative culture if the BD MAX™ Extended Enteric 28

Bacterial Panel and/ or other RT-PCRs were positive. Of the 251 samples, 200 were positive 29

and 51 were negative. Overall sensitivities of the Novodiag® Bacterial GE + kit for 30

Campylobacter sp, Salmonella sp, Shigella sp /EIEC, Yersinia enterocolitica, EHEC and 31

ETEC ranged from 98.98 to 100%, and specificities from 98.08 to 100%, PPV from 88.24 to 32

100% and NVP from 99.36 to 100%. 33

The analytical performance of the Novodiag® Bacterial GE+ kit is excellent. It can be used as 34

a routine tool in the rapid diagnosis of bacterial gastroenteritis. Despite the eNAT tube 35

dilution of the primary sample, the detection of Salmonella sp and EHEC was perfect. The kit 36

has the advantage of only detecting pathogenic Y. enterocolitica. Its performance for 37

Campylobacters is very satisfactory. 38

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Introduction 39

Intestinal infections continue to represent a major cause of morbidity and mortality 40

worldwide, with nearly 2 billion cases and 600,000 directly imputable deaths per year mostly 41

in children under the age of 5 years in developing countries (https://www.who.int/news-42

room/fact-sheets/detail/diarrhoeal-disease). Therefore, diagnosis of these infections must be 43

as quick and efficient as possible, especially since the diagnosis speed allows early 44

administration of targeted therapy. The bacterial infection diagnosis is traditionally based on 45

stool culture which is one of the least efficient techniques, due to a lack of sensitivity and a 46

tedious process requiring three days on average to obtain a final result for Campylobacter or 47

Salmonella species for example. Moreover, almost 80% of intestinal infection cases remain 48

undetected with this technique, partially due to a lack of detection of certain bacteria, e.g. 49

Escherichia coli pathovars (1, 2, 3). To overcome these limits, and with the technological 50

progress of the last decade, molecular biology techniques have been developed, including 51

syndromic multiplex panels which target several of the most common pathogens of enteric 52

infections. These multiplex assays also have the advantage to improve laboratory workflow 53

by reducing the analytical process (4). 54

Despite the theoretical advantages of syndromic panel-based assay use, questions remain 55

concerning its benefits and costs, which may vary among patient populations (4, 5, 6). 56

Furthermore, this strategy can become expensive if used on every stool samples without 57

considering the clinical and epidemiological context. Therefore, the implementation of a 58

syndromic panel-based strategy requires clinicians in association with microbiologists to 59

select cases for which a multiplex molecular assay would be useful, based on the Infectious 60

Disease Society of America (IDSA) or American College of Gastroenterology (ACG) 61

guidelines (7, 8, 9). Furthermore, the analytical performances and reliability of these panels 62

must be determined. Indeed, the clinical performance of these tests are generally evaluated 63

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internally by each of the companies that manufacture and market them. However, it is 64

important to assess its performance independently, by defining precisely the reference used 65

for each target and the clinical relevance of the results obtained. 66

Our aim was to evaluate the analytical performance of the Novodiag® Bacterial GE+ kit 67

(Mobidiag, Espoo, Finland), using 251 stool samples (198 retrospective and 53 prospective), 68

for the detection of enteropathogenic bacteria in acute community diarrhea. The analytical 69

performance was calculated using a composite reference standard (CRS). We showed that the 70

analytical performance of the Novodiag® Bacterial GE+ kit is excellent. It can be used as a 71

routine tool in the rapid diagnosis of bacterial gastroenteritis. 72

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Materials and Methods 73

Clinical specimens 74

Two hundred and fifty-one stool samples were included in this study (198 retrospective and 75

53 prospective). 76

The retrospective group was comprised of 1) 80 unpreserved stool specimens, collected 77

between April 2011 and January 2019 at the Pediatric and Adult Emergency Department of 78

the University Hospital Center of Bordeaux (Bordeaux, France), tested and positive by 79

conventional culture, and stored at -80°C within twelve hours after reception ; and 2) 118 80

stool specimens collected between November 2018 and March 2019, from three private 81

laboratories (Bio67, Strasbourg, France; CBM25, Besançon, France; and Exalab, Bordeaux, 82

France) and transported in Cary-Blair medium (Copan Diagnostics, Brescia, Italy). These 83

specimens in Cary-Blair medium were stored at -20°C within six hours after reception in the 84

respective laboratories and sent frozen to our laboratory. They were previously tested on-site 85

with the BD MAX™ Extended Enteric Bacterial Panel (which detects Campylobacter jejuni 86

and Campylobacter coli, Salmonella sp, Shigella /enteroinvasive E. coli (EIEC), Yersinia 87

enterocolitica, Plesiomonas shigelloides, Vibrio sp, entero-haemorrhagic E. coli (EHEC) and 88

enterotoxigenic E. coli (ETEC)) and by culture: 116 of these 118 samples were positive 89

(according to BD MAX™ Extended Enteric Bacterial Panel result (n=118) and/or culture 90

(n=87)) for at least one enteropathogen. The results obtained on these 198 stool samples are 91

shown in Table 1. 92

The prospective group was comprised of 53 unpreserved stool samples received from the 93

Pediatric and Adult Emergency Departments of Bordeaux University Hospital (Table 1), from 94

February to April 2019, and tested with conventional culture, stored at +4°C and analyzed 95

with the Novodiag® Bacterial GE+ within 2 days. Only 5 of them were positive by 96

conventional culture (Table 1). 97

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None of these 251 stools underwent other freeze–thaw cycles. 98

99

Routine methods for bacterial culture 100

Stool specimens from Bordeaux hospital were tested for bacterial enteropathogens using 101

conventional culture methods. They were plated on Hektoen Enteric (bioMérieux, Marcy 102

l’Étoile, France), Campylosel (bioMérieux) and Cefsulodin-Irgasan-Novobiocin (CIN) 103

(Oxoid, Basingstoke, UK) media. Additionally, a Selenite-Lactose broth (ThermoFischer 104

Scientific, Waltham, MA) was inoculated, incubated overnight at +35°C, and used to 105

inoculate a second Hektoen Enteric plate after enrichment. All plates were incubated two days 106

at +35°C in ambient air, except for Campylosel, which was incubated three days in jars using 107

an Anoxomat microprocessor (Mart Microbiology, B.V. Lichtenvoorde, The Netherlands) 108

which creates a microaerobic atmosphere (80–90% N2, 5–10% CO2, and 5–10% H2), and for 109

CIN which was incubated at +30°C. Bacterial identification was performed by matrix-assisted 110

laser desorption ionization–time of flight mass spectrometry (Bruker Daltonics, Bremen, 111

Germany), as previously described (10). 112

113

The 118 retrospective samples were plated on a selective medium based to the bacterium 114

detected by BD MAX™ Extended Enteric Bacterial Panel (CIN, Campylosel or Hektoen 115

Enteric): 31 of them were negative for Campylobacter culture despite a positive result 116

obtained on BD MAX™ Extended Enteric Bacterial Panel. The Campylobacter status of these 117

31 samples had been confirmed in a previous study with another PCR format, i.e., the PCR 118

Rida®Gene Bacterial Stool Panel (R-Biopharm AG, Darmstadt, Germany) (11). 119

Table 1 summarizes the bacterial status of the samples according to the diagnostic tests 120

performed on these 251 samples. 121

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122

Novodiag® Bacterial GE+ testing 123

The Novodiag® Bacterial GE+ assay was performed using a diluted sample in eNATTM

124

medium (Copan Diagnostics). The dilution was carried out by introducing faeces into a tube 125

using a FLOQ Swab (Copan Diagnostics), or 300µL of Cary-Blair medium. This tube 126

contains a solution which inactivates infectious agents by chemical lysis within 30 min and 127

stabilizes genomic material by nuclease inactivation. Six hundred µL of eNATTM

solution was 128

then transferred to the Novodiag® Bacterial GE+ cartridge, according to the manufacturer’s 129

instructions, and put in the Novodiag® system. 130

The Novodiag® Bacterial GE+ test is based on automated nucleic acid extraction, 131

amplification and analysis with two technologies: real-time PCR (fluorescent probes) and 132

microarray (total internal reflection fluorescence (TIRF)-based detection). The test is 133

CE/IVD- marked, as well as the instrument. The total duration of the process (sample and 134

reagents preparation, cassette incubation and PCR) takes around 105 min (1.75h). Four 135

modules of 4 cassettes can be connected to the Novodiag® system. The Novodiag® software 136

automatically performs an analysis with each target reported as positive or negative, with the 137

list of detected targets (Table S1). If a control fails, Novodiag® reports the run as invalid for 138

every target. According to the manufacturer’s instructions, every sample with an invalid result 139

was retested a second time and, if needed, a third time with a diluted sample (300µL of the 140

first eNATTM

tube transferred to a second eNATTM

). These retests were processed within 24 141

hours of the first result. 142

143

Confirmation method 144

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Samples showing discordant results between Novodiag® Bacterial GE+ and culture and/or 145

expected BD MAX™ Extended Enteric Bacterial Panel PCR results were further analyzed by 146

an additional confirmation test (Table 2). Every sample with a discordant positive result 147

concerning Campylobacter sp or Salmonella sp with the Novodiag® Bacterial GE+ was 148

confirmed with the PCR Rida®Gene Bacterial Stool Panel (R-Biopharm) as previously 149

described (11). 150

Every discordant result for E. coli pathovars detected by Novodiag® Bacterial GE+ was 151

confirmed by endpoint PCR for specific virulence-associated genes: ipaH/ial for Shigella 152

/EIEC, lt/stp for ETEC, aggR for enteroaggregative E. coli (EAEC), eae/bfpA for 153

enteropathogenic E. coli (EPEC) using the primers and PCR conditions described by Oh et 154

al., (12), and stx1 and stx2 for STEC using the primers and PCR conditions described by Grad 155

et al. (13). The PCR reactions contained 2.5 µl of prepared genomic DNA, 5µL Buffer MgCl2 156

5x (Promega, Madison, WI, USA), 0.5µL of dNTPs (10mM, Promega), 0.1µL of each primer 157

(100µM, Eurofins Genomics, Ebersberg, Germany), 0.3µL of GoTaq® DNA polymerase 158

(5UI/µL, Promega) and water to a final volume of 25µl. PCRs were carried out on a 159

Mastercycler® Nexus (Eppendorf, Hamburg, Germany) under the following conditions: an 160

initial denaturation step at 94°C for 4 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 161

30 sec and 72°C for 30 sec, followed by a final extension of 72°C for 7 min. Amplified DNA 162

products were loaded onto a 1-3% agarose gel containing SYBRTM

Safe (Fisher Scientific, 163

Illkirch, France) and read under UV using the Genflash system (SynGene, BioImaging, 164

Firesdrick, MD, USA). Concerning EHEC detection, an overnight enrichment of the samples 165

in brain-heart broth (Oxoid) was performed, as recommended by Gouali et al., (14) followed 166

by DNA extraction with MagNA Pure 96 (Roche Diagnostic, Meylan, France) followed by 167

stx1 and stx2 PCR detection. Finally, every Clostridioides difficile detection was confirmed 168

with Alethia® C. difficile (Meridian Biosciences, Cincinnati, OH, USA). The Alethia® C. 169

difficile assay utilizes a loop-mediated isothermal DNA amplification (LAMP) technology to 170

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detect the pathogenicity locus (PaLoc) of toxigenic C. difficile which codes for both the Toxin 171

A gene (tcdA) and the Toxin B gene (tcdB) (15). 172

All Y. enterocolitica isolates were sent to the National Reference Center of yersiniosis 173

(Pasteur Institute, Paris, France) for biotyping. 174

175

Population and clinical study 176

The median age of the 251 patients was 30.4 years (+/- 28.2) with a sex ratio of 1.19. Clinical 177

data was collected for the patients hospitalized in Bordeaux University Hospital (Bordeaux 178

CHU) included in the retrospective stool group (n = 74) and for all patients who were part of 179

the prospective stool group (n = 53). The median age of this population was 22.2 years (+/- 180

27.1) and the sex ratio was 1.25. 181

The clinical features collected were: presence of diarrhea, fever, abdominal pain, rectal 182

bleeding, nausea and vomiting. In addition, information on the initiation of a probabilistic 183

antibiotic therapy or oral rehydration, the length of hospitalization and the presence of a 184

biological inflammatory syndrome (CRP > 5 mg/L) were obtained. 185

186

Statistical analysis and composite reference standard (CRS) 187

The analytical performances were calculated using a composite reference standard (CRS). 188

The CRS was defined as positive when culture was positive or, in the case of a negative 189

culture (or if conventional culture was not performed or unable to detect the pathogen, ie., 190

EHEC, EPEC, EAEC, ETEC, C. difficile), when the result was concordant with the one 191

previously obtained with the BD MAX™ Extended Enteric Bacterial Panel. When a 192

discrepancy was noted, the CRS was defined as positive when at least one independent PCR 193

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assay used for confirmation was concordant with the Novodiag® Bacterial GE+ (Table 2). 194

The 95% confidence interval was calculated using the Wilson method. 195

This CRS was applied for Campylobacter sp, Salmonella sp, Shigella sp /EIEC, Y. 196

enterocolitica, EHEC and ETEC. EAEC and EPEC were excluded because of the absence of 197

a culture result and they were not included in the BD MAX™ Extended Enteric Bacterial 198

Panel. C. difficile was also not included in the BD MAX™ Extended Enteric Bacterial Panel. 199

For these 3 pathogens only concordance percentages between Novodiag® Bacterial GE+ 200

results and confirmation PCRs were calculated. 201

To compare clinical features, a Chi-square or F-test were performed, with a p value <0.05 202

considered as significant. 203

204

Ethics 205

All diagnostic methods were performed routinely. All patients were investigated in a hospital 206

or private setting, according to good clinical practices. No informed consent for using human 207

stool samples was requested of the patients. Therefore, to ensure subject anonymity, all 208

indirectly identifiable patient data were removed from the present study. 209

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Results 210

211

Retrospective study 212

One hundred ninety-eight stool samples were included in the retrospective study (Table 1). 213

Novodiag® Bacterial GE+ detected the presence of Campylobacter sp in 89 of the 90 positive 214

samples. The negative sample corresponded to a C. jejuni case for which the culture was 215

negative. The presence of C. jejuni was confirmed by a Rida®Gene Bacterial Stool Panel 216

both on the primary sample and the eNAT tube. Novodiag® Bacterial GE+ correctly detected 217

the samples positive for Shigella sp (n=7) and for Salmonella sp (n=45). Furthermore, 218

Novodiag® detected 7 Y. enterocolitica among 9 stools positive by culture. The 2 Y. 219

enterocolitica not detected by Novodiag® Bacterial GE+ belonged to the non-pathogenic 220

biotype 1a, as confirmed by the NRC for yersiniosis (data not shown). Novodiag® Bacterial 221

GE+ also detected 26 EHEC cases (19 EHEC alone, 7 in combination with another 222

enteropathogen: 3 with C. jejuni, 3 with ETEC, 1 with Salmonella sp). Novodiag® Bacterial 223

GE+ was negative for one sample that was expected to be positive for EHEC according to the 224

results previously obtained on the BD MAX™ Extended Enteric Bacterial Panel. The 225

presence of EHEC was not confirmed by end-point stx1 and stx2 PCR. Novodiag® Bacterial 226

GE+ detected 17 ETECs (9 ETEC alone, 8 in combination with 1 to 2 other enteropathogens: 227

2 with EPEC, 1 with C. jejuni, 1 with C. jejuni + EAEC, 3 with EHEC, 1 with EAEC). 228

Finally, 3 C. difficile (2 with Salmonella sp, 1 with C. jejuni), 14 EPECs (3 EPEC alone, and 229

11 with 1 to 3 other enteropathogens) and 9 EAECs (1 EAEC alone and 8 in combination 230

with 1 to 3 other enteropathogens) were also detected by Novodiag®: the presence of C. 231

difficile in the 3 stool samples was confirmed by Alethia® C. difficile, as well as 11 EPEC 232

(78.6%) and 5 EAEC (55.6%) by end point PCR (Table 3). 233

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Concerning the 13 stools that were expected to be negative, Novodiag® Bacterial GE+ 234

detected 1 EAEC and 1 EPEC which were not confirmed by end-point PCR. 235

Prospective study 236

Among the 53 prospective stool samples, 18 cases (34%) were positive for one or several 237

enteric pathogens using Novodiag® Bacterial GE+, whereas only 5 cases (9.4%) (2 C. jejuni, 238

1 C. coli, 1 Salmonella sp, 1 Y. enterocolitica) were positive by conventional culture. All 239

bacteria species identified by culture were also detected using Novodiag® Bacterial GE+. 240

Novodiag® Bacterial GE+ detected 4 pathogens that should have been found by conventional 241

culture: 2 C. jejuni (confirmed with PCR Rida®Gene Bacterial Stool Panel) (1 with EPEC as 242

confirmed by end-point PCR) and 2 Shigella /EIEC (confirmed by ipaH and ial PCR). 243

Novodiag® Bacterial GE+ also detected 3 C. difficile (confirmed with Alethia® C. difficile), 244

2 EHEC (both confirmed by stx1/stx2 PCR), 2 EPEC alone (1 confirmed) and 1 EPEC+EAEC 245

(EPEC confirmed only) (Table 3). 246

247

Global study 248

The analytical process went smoothly for most of the samples. “Invalid” results were obtained 249

on 9 eNATTM

samples: four contained important traces of blood and three were too heavily 250

loaded with stools, both situations being conductive to the inhibition of PCR. These problems 251

were solved after retesting (see material and methods). The results for all of the samples 252

included in the present study are summarized in Table 3: 62 of the 251 samples (24.7%) were 253

expected to be negative (based on culture and BD MAX™ Extended Enteric Bacterial Panel 254

screening). The 3 main pathogens that comprised this set of samples were: first 255

Campylobacter sp (93/251, 37.1%), followed by Salmonella sp (46/251, 18.3%) and 256

pathogenic E. coli (37/251, 14.7%). After PCR confirmation of the discordant results obtained 257

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using the Novodiag® Bacterial GE+ kit, 51 of the 251 remained negative (20.3%), 200 of the 258

251 were therefore positive (79.7%). Among the 200 positive, 178 were positive for one 259

single pathogen (88.6%), and 22 in combination with 1 to 4 other pathogens (11.4%) (Table 260

3). Novodiag® Bacterial GE+ additionally detected 32 Campylobacter, 14 EHEC and 2 261

Shigella /EIEC compared to culture only and detected only pathogenic Y. enterocolitica. 262

Considering the CRS defined for the present study (see Materials and Methods), Novodiag® 263

Bacterial GE+ sensitivity ranged from 98.98% for Campylobacter to 100% for the other major 264

enteric pathogens. The specificity fluctuated from 98.08% for ETEC to 100% (Table 4A-B). 265

Overall agreement (EPEC, EAEC and C. difficile excluded) was 98.80% (96.54 - 99.59). 266

Concordant percentages between Novodiag ®Bacterial GE+ were: 100% for C. difficile (all 6 267

cases confirmed), 72.2% for EPEC (13 out of 18 cases confirmed) and 50% for EAEC (5 out 268

of 10 cases confirmed). 269

270

Demographics and clinical features 271

The demographic characteristics associated with 128 specimens from patients hospitalized in 272

emergency units of the Bordeaux CHU (74 included in the retrospective stool collection) and 273

all patients who were part of the prospective stool collection(n = 53)) are presented in Table 274

5. Those with positive results with Novodiag® Bacterial GE+ (confirmed in case of 275

discordance) (92/127, 72.4%) had significantly more fever, diarrhea, abdominal pain or blood 276

in their feces. Moreover, these patients received probabilistic antibiotic therapy more often 277

(Table 5). In the positive group, the median age was 17 years (+/- 21.6) with a sex ratio of 278

1.21. In the negative group, the median age was 36.2 years (+/- 34.2) with a sex ratio of 1.33. 279

Among the 93 patients with positive results with Novodiag® Bacterial GE+, 11 were positive 280

for a bacterium not detectable by conventional co-culture: 4 EPEC, 1 EAEC, 1 EAEC + 281

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EPEC, 2 EHEC, and 3 toxinogenic B C. difficile. These patients showed characteristics 282

similar to those of other patients in the positive group (data not shown). 283

Discussion 284

The diagnosis of acute digestive bacterial infections must be as rapid and precise as possible, 285

since a rapid diagnosis conditions the establishment of a targeted therapy at an early stage. 286

Nevertheless, these infections are often under-documented because conventional diagnostic 287

techniques are long, tedious and require qualified personnel. The use of syndromic PCR 288

panels reduces the mass of analytical work. However, it is necessary to evaluate the analytical 289

performances and the reliability of the resulting diagnosis of the various existing kits. The 290

present study aimed at evaluating the performance of the Novodiag® Bacterial GE+ kit. 291

This study shows that the Novodiag® Bacterial GE+ kit has an excellent analytical 292

performance to detect bacterial enteropathogens in stools. This new kit showed a better 293

sensitivity than culture in accordance to the literature on syndromic molecular diagnosis (15, 294

16, 17, 18, 19, 20, 21, 22, 23, 24). This gain of sensitivity allowed the detection of 2 Shigella 295

/EIEC and 2 C. jejuni from patients with negative culture but suggestive symptoms. 296

Furthermore, Novodiag® Bacterial GE+ enabled the detection of the major E. coli pathovars 297

and, in our study, Novodiag® Bacterial GE+ performed even better that the BD 298

MAX™system (25), in particular for ETEC and STEC detection. Novodiag® Bacterial GE+ 299

detects only the pathogenic biotype of Y. enterocolitica, which highlights the rational of the 300

target gene used in this kit. The detection of the major bacterial enteropathogens involved in 301

community-acquired human digestive infections is excellent. The absence of appropriate CRS 302

and the low number of EAEC and EPEC cases did not allow us to properly assess the 303

performance for these pathogens. Nor could we evaluate C. difficile which was also beyond 304

the scope of our study, which focused on cases of acute community-acquired digestive 305

infections. If it is deemed necessary, more specialized and targeted studies will allow a future 306

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evaluation of this kit regarding these pathogens. Finally, the analysis of clinical features of a 307

set of the patients enrolled in the present study showed the clinical relevance of the 308

Novodiag® Bacterial GE+ results. 309

In the present study one false negative C. jejuni infection was found. The corresponding 310

sample was positive using the Rida®Gene Bacterial Stool Panel with a Ct:31.1 on the primary 311

specimen, and 43.6 on the eNAT tube. This deviation corresponds to a 5,000-fold dilution, 312

while theoretically it should not exceed 8-fold, thus revealing an over-dilution due to a pre-313

analytical error. Accordingly, the dilution in the eNAT tube could in theory be associated with 314

a loss of sensitivity and punctually the risk of obtaining false negative results by over-315

dilution. It reveals the importance of mastering pre-analytical conditions to obtain 316

interpretable and reliable results. The Novodiag® system does not provide amplification 317

curves (nor Ct values) or fluorescence intensity values which could help microbiologists to 318

interpret the results as previously suggested (11), in particular in line with the bacterial 319

inoculum. This also complicates interpretation of the results, particularly in the event of co-320

infections (11.4% in the present study after confirmation). 321

The Novodiag® Bacterial GE+ can detect EHEC based on the detection of specific target 322

genes: eae, stx1 and stx2. EHEC infections are sometimes associated with the development of 323

hemolytic uremic syndrome. In France EHEC strains have a known gene arsenal comprised of 324

eae+ stx2 in 60% of cases (data from the NRC for E. coli, Pasteur Institute, France; http://cnr-325

escherichiacoli-robertdebre.aphp.fr/). However, this profile was found in 2 cases included in 326

the present study. To our knowledge, no EHEC positive patient developed hemolytic uremic 327

syndrome. Furthermore, in 46.4% of these cases the Novodiag® detected stx1 only. These 328

different elements illustrate the need for interpretation of these results by the microbiologist, 329

in sync with the clinician. 330

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The Novodiag® Bacterial GE+ kit allows a reliable result within only 2 hours. The use of this 331

type of multiplex PCR implies the exclusive detection of the targets of the proposed panel. 332

Campylobacter species other than C. jejuni and C. coli, Arcobacter sp, Aeromonas sp, and 333

Plesiomonas shigelloides are undetectable with Novodiag® Bacterial GE+. This observation 334

must also be taken into account when developing the strategy for using this kit, in particular 335

with regard to the choice of hospital departments which can prescribe this analysis. 336

Despite the theoretical advantages of the diagnostic panels compared to standard techniques, 337

their positive consequences on patient care and their economic impact have not been clearly 338

objectified and are extremely variable depending on the populations studied (4, 5, 6, 25, 26, 339

27, 28). With a view to the putative implementation of the Novodiag® Bacterial GE+ kit at 340

the Bordeaux University Hospital, this study allowed us to target the patient profile for which 341

there would be a benefit. Due to the Novodiag® research panel, as discussed above, it is not 342

intended for all hospitalized patients: the aim of the Novodiag® Bacterial GE+ kit is to detect 343

the major pathogens responsible for acute gastroenteritis syndromes. In this case, it is of 344

particular interest in community-acquired infections. According to internal data for the year 345

2018 (not shown) from the Bordeaux University Hospital, 74% of the enteropathogenic 346

bacteria identifiable by both Novodiag® Bacterial GE+ and culture, with the exception of C. 347

difficile, came from adult and pediatric emergency and post-emergency wards. Therefore the 348

implementation of the Novodiag® technology or any other syndromic PCR in our hospital 349

would be more appropriate in terms of benefit for emergency and post-emergency wards 350

especially on all bloody and watery stools. An oriented stool culture strategy, following the 351

results of Novodiag®, could be an option for the samples coming from these units. 352

In conclusion, our study demonstrates the excellent technical performance of the Novodiag® 353

kit. This study could serve as a basis for others for the implementation of a syndromic 354

molecular biology technology in terms of workflow adaptation between the prescribing 355

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services and the laboratory, all participating, for the above-mentioned reasons, in a necessary 356

dialogue between the biologist and the clinician for an appropriate use and interpretation, all 357

in the best interest of the patient. 358

359

Acknowledgment 360

We thank Mrs. Lindsay Mégraud, a native English speaker, who carefully reviewed this 361

paper. We also sincerely thank Dr. T Gueudet (Bio-67, Strasbourg, France) and Dr. MC 362

Paolini (CBM25, Besançon, France) who provided stools samples included in the present 363

study. 364

365

Conflict of interest 366

The authors declare no conflict of interest. Mobidiag company (Espoo, Finland) which 367

commercializes the Novodiag® Bacterial GE+ test in France provided the kits but was not 368

involved in the study design nor in the data analysis. 369

370

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Table 1. Stool samples used in the study 1

2

Source Expected bacterial status BDMax Culture

Retrospective

samples (n=198)

Bordeaux University

Hospital (n=80)

40 C. jejuni nt X

18 Salmonella sp nt X

1 Salmonella sp + C. difficile nt X (pos for both)

3 C. jejuni + C. coli nt X

2 C. coli nt X

3 Shigella sp nt X

2 Y. enterocolitica nt X

11 Negative nt X

Private laboratories

(n=118)

39 C. jejuni X X (26 neg)

3 C. coli X X (3 neg)

26 Salmonella sp X X

22 EHEC X nt

13 ETEC X nt

6 Y. enterocolitica X X

4 Shigella sp X X

1 C. jejuni + EHEC X X (neg for both)

1 C. jejuni + ETEC X X (neg for both)

1 Y. enterocolitica + C. jejuni X X

2 Negative X X

Prospective samples

(n=53)

2 C. jejuni nt X

1 C. coli nt X

1 Salmonella sp nt X

1 Y. enterocolitica nt X

48 Negative nt X

X: done; nt: not tested; pos: positive; neg: negative. BDMax = BD MAX™ Extended Enteric 3

Bacterial Panel. EHEC: entero-haemorrhagic E. coli ; ETEC: enterotoxigenic E. coli. 4

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Table 2. Test considered for the determination of the initial bacterial status of the samples 1

and confirmation methods used in the study 2

3

Bacteria detected by

Novodiag® Bacterial GE+

Test considered for the

determination of the initial

bacterial status

Confirmation method if

necessary

Campylobacter coli Positive culture or

PCR BD MAX™ positive

Rida®Gene Bacterial Stool

Panel

Campylobacter jejuni Positive culture or

PCR BD MAX™ positive

Rida®Gene Bacterial Stool

Panel

Salmonella sp Positive culture or

PCR BD MAX™ positive

Rida®Gene Bacterial Stool

Panel

Shigella sp /EIEC Positive culture or

PCR BD MAX™ positive

ipaH and ial PCR

Yersinia enterocolitica Positive culture biotyping*

EHEC PCR BD MAX™ positive stx1 and stx2 PCR

ETEC PCR BD MAX™ positive lt and stp PCR

Clostridioides difficile none Alethia C. difficile

EAEC none aggR PCR

EPEC none bfpA and eae PCR

*biotyping performed at the National Reference Center for yersiniosis 4

BD MAX™ = BD MAX™ Extended Enteric Bacterial Panel. 5

EPEC: enteropathogenic E. coli; EHEC: entero-haemorrhagic E. coli; ETEC: enterotoxigenic 6

E. coli; EIEC: enteroinvasive E. coli; EAEC: enteroaggregative E. coli. 7

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Table 3. Comparison between the expected results, those obtained using Novodiag® Bacterial GE+ and after confirmation. 1

2

Expected result (No.) R P Positive

culture

Novodiag®Bacterial GE+

result

Confirmation Not

confirmed

Negative (61) 13 48 0 45 neg

1 C. jejuni 1 C. jejuni

1 C. jejuni+ EPEC 1 C. jejuni + EPEC

2 Shigella sp /EIEC 2 Shigella sp /EIEC

3 C. difficile 3 C. difficile

1 EAEC 1 EAEC

5 EPEC 2 EPEC 3 EPEC

2 EHEC 2 EHEC

1 EAEC + EPEC 1 EPEC 1 EAEC

C. jejuni (81) 79 2 54 69 C. jejuni

1 C. jejuni + C. difficile 1 C. difficile

2 C. jejuni + EAEC + EPEC 2 EPEC + 1 EAEC 1 EAEC

2 C. jejuni + EHEC 2 EHEC

5 C. jejuni + EPEC 3 EPEC 2 EPEC

1 C. jejuni + ETEC 1 ETEC

1 neg 1 C. jejuni

C. jejuni + EHEC (1) 1 0 0 1C. jejuni + EHEC

C. jejuni + ETEC (1) 1 0 0 1 C. jejuni + ETEC + EAEC 1 EAEC

C. coli (6) 5 1 3 6 C. coli

C. jejuni + C. coli (3) 3 0 3 (for both) 1 C. jejuni + C. coli

1 C. jejuni + C. coli + EAEC 1 EAEC

1 C. jejuni + C. coli + EAEC +

EPEC

1 EAEC + 1 EPEC

Shigella sp (7) 7 0 7 7 Shigella sp

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Y. enterocolitica (9) 8 1 9 7 Y. enterocolitica

1 Y. enterocolitica + EPEC 1 EPEC

1 neg 1 non pathogenic Y.

enterocolitica

Y. enterocolitica + C. jejuni (1) 1 0 1 (for both) 1 C. jejuni 1 non pathogenic Y.

enterocolitica

EHEC (22) 22 0 nd 19 EHEC

2 EHEC + ETEC 1 ETEC 1 ETEC

1 NEG 1 NEG

ETEC (13) 13 0 nd 9 ETEC

2 ETEC + EPEC 2 EPEC

1 ETEC + EHEC 1 EHEC

1 ETEC + EAEC 1 EAEC

Salmonella sp (45) 44 1 45 41 Salmonella sp

1 Salmonella sp + C. difficile 1 C. difficile

2 Salmonella sp + EAEC 2 EAEC

1 Salmonella sp + EHEC 1 EHEC

Salmonella sp + C. difficile (1) 1 0 1* 1 Salmonella sp + C. difficile

198 53 135

nd : not done ; *for Salmonella sp only 3

In bold discordant result obtained by Novodiag® Bacterial GE+ compared to expected result. 4

R: retrospective study; P: prospective study. 5

EPEC: enteropathogenic E. coli; EHEC: entero-haemorrhagic E. coli; ETEC: enterotoxigenic E. coli; EIEC: enteroinvasive E. coli; EAEC: 6

enteroaggregative E. coli. 7

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Table 4. Performance of the Novodiag® Bacterial GE+. 1

(4A) 2

Composite reference standard (No. of results)

Pathogen (No. of samples) Results Positive Negative Total

Campylobacter sp (251) Positive 94 0 94

Negative 1 156 157

Salmonella sp (251) Positive 46 0 46

Negative 0 195 195

EHEC (123)* Positive 28 0 28

Negative 0 95 95

ETEC (119) Positive 15 2 17

Negative 0 102 102

Y. enterocolitica (251) Positive 8 0 8

Negative 0 243 243

Shigella sp /EIEC (251) Positive 9 0 9

Negative 0 242 242

The CRS was defined as positive when culture was positive or, in the case of a negative culture (or if conventional culture was not performed or 3

unable to detect the pathogen (i.e., EHEC, EPEC, EAEC, ETEC, C. difficile)), when the result was concordant with the one previously obtained 4

with the BD MAX™ Extended Enteric Bacterial Panel. When a discrepancy was noticed, CRS was defined as positive when at least one 5

independent PCR assay used for confirmation was concordant with Novodiag® Bacterial GE+. 95% confidence intervals were calculated using 6

the Wilson method. 7

(4B) 8

Campylobacter sp EHEC Shigella sp /EIEC ETEC Salmonella sp Y. enterocolitica

Sensitivity 98.98 (94.45 - 99.82) 100 (84.98-100) 100 (62.88 - 100) 100 (74.65 - 100) 100 (92.29 - 100) 100 (59.77 - 100)

Specificity 100 (97.60 - 100) 100 (96.11 -100) 100 (98.44 - 100) 98.08 (93.26–99.47) 100 (98.07 - 100) 100 (98.44 - 100)

PPV 100 (96.07 - 100) 100 (84.98-100) 100 (62.88 - 100) 88.24 (62.26–97.94) 100 (92.29 - 100) 100 (59.77 - 100)

NPV 99.36 (96.48 -99.89) 100 (96.11 -100) 100 (98.44 - 100) 100 (96.37- 100) 100 (98.07 - 100) 100 (98.44 - 100)

PPV: positive predictive value; NPV: negative predictive value. EHEC: entero-haemorrhagic E. coli; ETEC: enterotoxigenic E. coli; EIEC: 9

enteroinvasive E. coli. 10

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Table 5. Clinical features of Bordeaux University Hospital patients. 1

1data available for 33 patients;

2data available for 84 patients 2

*: p<0.05 ; **: p <0.001 3

Symptoms Negative group

(n=35)

(95% CI)

Positive group

(n=92)

(95%CI)

p

Diarrhea 68.6 (52.0 – 81.5) 98.9 (94.1 – 99.8) < 0.001**

Vomiting 34.3 (20.8 – 50.9) 41.3 (31.8 – 51.5) 0.464

Bloody stools 8.6 (3.0 – 22.4) 44.6 (34,9 – 54.8) < 0.001**

Fever 34.3 (20.8 – 50.9) 72.8 (62.9 – 80.8) < 0.001 **

Biological inflammatory

syndrome

60.6 (43.7 – 75.3)1 58.3 (47.6 – 68.3)

2 0.835

Rehydration 11.4 (4.5 – 25.9) 20.7 (13.7 – 30.1) 0.235

Probabilistic

antibiotherapy

37.2 (23.2 – 53.7) 57.6 (47.4 – 67,2) 0.038*

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